Methods for detecting a target polynucleotide in a sample

Abstract

Disclosed are methods for detecting a target polynucleotide comprising a genetic alteration relative to a reference polynucleotide in a sample comprising a mixture of the target polynucleotide and the reference polynucleotide. The methods may be utilized for detecting with high selectivity a target polynucleotide in a mixture of the reference polynucleotide and the target polynucleotide in which the target polynucleotide represents a low percentage of the mixture. The methods may be adapted for diagnosing, prognosing, and treating subjects having a disease or disorder associated with the genetic alteration.

1 . A method for detecting a target polynucleotide comprising a genetic alteration relative to a reference polynucleotide lacking the genetic alteration in a sample comprising a mixture of the target polynucleotide and the reference polynucleotide, the method comprising:
 (a) performing a polymerase chain reaction (PCR) amplification to obtain an amplification product in a reaction mixture comprising:
 (i) the sample; 
 (ii) a high fidelity DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity; 
 (iii) a pair of primers that flank the genetic alteration; and 
 (iv) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex; and 
   (b) sequencing the amplification product and detecting the genetic alteration in the amplification product, thereby detecting the target polynucleotide comprising the genetic alteration in the sample.   
     
     
         2 . The method of  claim 1 , wherein the target polynucleotide represents no more than about 3% of the mixture. 
     
     
         3 . The method of  claim 1 or 2 , wherein the target polynucleotide is present in the sample at a copy number less than about 30. 
     
     
         4 . The method of  claim 1 , wherein the PCR amplification is performed for no more than 60 cycles. 
     
     
         5 . The method of  claim 1 , wherein the DNA polymerase has an error rate of less than 10 −5 . 
     
     
         6 . The method of  claim 1 , the amplification product is sequenced to obtain multiple reads, and the genetic alteration is present in at least about 90% of the multiple reads. 
     
     
         7 . The method of  claim 1 , wherein the genetic alteration is present in at least about 90% of the amplification product. 
     
     
         8 . The method of  claim 1 , the method comprising removing the blocking oligonucleotide from the amplification product prior to sequencing the amplification product. 
     
     
         9 . The method of  claim 1 , further comprising determining the minimum number of PCR cycles necessary for detecting a target polynucleotide that is present in the mixture at a percentage of no more than 0.1%, 0.05%, 0.02%, or 0.01%. 
     
     
         10 . The method of  claim 1 , wherein the blocking oligonucleotide comprises one or more modified nucleotides that enhance the stability of the duplex formed by the blocking oligonucleotide and the reference polynucleotide. 
     
     
         11 . The method of  claim 1 , wherein the blocking oligonucleotide comprises one or more modified nucleotides that enhance the selectivity of the blocking oligonucleotide for hybridizing to the reference polynucleotide relative to the target polynucleotide. 
     
     
         12 . The method of  claim 1 , wherein the blocking oligonucleotide comprises one or more locked nucleic acids (LNAs) comprising a sugar moiety having a 2′-O conjugated to 4′-C via a methylene group. 
     
     
         13 . The method of  claim 12 , wherein all of the nucleotides of the blocking oligonucleotide are LNAs. 
     
     
         14 . The method of  claim 1 , wherein the blocking oligonucleotide has a length that is no more than about 20 nucleotides. 
     
     
         15 . The method of  claim 1 , wherein the genetic alteration comprises a substitution. 
     
     
         16 . The method of  claim 1 , wherein the genetic alteration comprises a deletion. 
     
     
         17 . The method of  claim 1 , wherein the genetic alteration comprises an insertion. 
     
     
         18 . The method of  claim 1 , wherein the genetic alteration is present in a genetic loci selected from the group consisting of PIK3CA, KRAS, APC, FAT4, KMT2D, KMT2C, and BRAF. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein the sample is a biological sample from a subject. 
     
     
         21 . The method of  claim 1 , wherein the sample is a blood sample from a subject. 
     
     
         22 . The method of  claim 21 , wherein the blood sample is a cell-free blood sample and/or the blood sample is treated to remove cells prior to performing PCR amplification. 
     
     
         23 . The method of  claim 1 , wherein prior to the method being performed the sample was treated with a reagent that selectively modifies non-methylated cytosine residues to produce detectable modified residues but which does not modify methylated cytosine residues. 
     
     
         24 . The method of  claim 1 , wherein the genetic alteration is associated with cancer in a subject. 
     
     
         25 . The method of  claim 24 , wherein prior to performing step (a), the method comprises sequencing a cancer sample from the subject and detecting the genetic alteration in the cancer sample. 
     
     
         26 . The method of  claim 1 , wherein the reference polynucleotide comprises a wild-type sequence with respect to the genetic alteration and the genetic alteration is a genetic mutation present in the target polynucleotide. 
     
     
         27 . The method of  claim 1 , wherein the sample is obtained from a subject who has cancer or is at risk for developing cancer. 
     
     
         28 . The method of  claim 1 , wherein the subject has been diagnosed with cancer and the subject currently is in remission. 
     
     
         29 . (canceled) 
     
     
         30 . A kit for performing a method for detecting a target polynucleotide comprising a genetic alteration relative to a reference polynucleotide lacking the genetic alteration in a sample comprising a mixture of the target polynucleotide and the reference polynucleotide, wherein the kit comprises: (i) a DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity; (ii) a pair of primers that flank the genetic alteration; and (iii) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex and obtaining an amplified fragment sample. 
     
     
         31 . The kit of  claim 30 , wherein the genetic alteration is in a genetic loci selected from PIK3CA, KRAS, APC, FAT4, KMT2D, KMT2C, and BRAF. 
     
     
         32 . The kit of  claim 30 , wherein the blocking oligonucleotide consists of LNAs and the blocking oligonucleotide is no longer than 20 nucleotides.